The objective of the proposed research is to study the organization of glycosphingolipids in liposomal systems and in plasma membranes of mammalian cells. Morphological techniques for visualization of glycosphingolipids will be utilized, including localization by freeze-Atch electron microscopy of ferritin-conjugates of lectins and antibodies with activity directed against specific glycosphingolipids. Morphological findings will be correlated with biophysical studies of glycosphingo-lipid organization, using techniques of differential scanning calorimetry, NMR, and fluorescence polarization. The organization of glycosphingolipids will be evaluated in liposomes consisting of varying mixtures of phospholipids, cholesterol, and glycosphingolipids, with comparison to liposomes containing purified glycoproteins such as red blood cell Band 3 and PAS I glycoproteins and purified sarcoplasmic reticulum CA2ion ATPase. Ferritin conjugated anti-glycosphingolipid markers will be used to evaluate whether the glycosphingolipids are randomly distributed in the liposomes, or whether they are organized into clusters or domains, and also whether they show any structural relationship to proteins incorporated into the lipid bilayers. The ferritin-conjugated reagents will also be used to evaluate the distribution of glycosphingolipids in the red blood cell membranes of various species and in normal and transformed cells.